The ultimate expression of the genetic makeup of a cell resides in the proteins of that cell. Although protein synthesis is a complex process requiring numerous proteins and nucleic acids, to a large extent the specificity is determined by the first step of the process - the activation and transfer of an amino acid to its respective transfer RNA. Although considerable information about aminoacyl-tRNA synthetases and TRNA has been obtained, relatively little is known about the control of protein synthesis that may be exercised at the level of aminoacyl-tRNA formation. Our long term goal is the elucidation of the pathways, enzymes and regulatory factors which determine the levels of tRNA and synthetases in cells, and their effect on the various cellular processes in which aminoacyl-tRNA participates. The studies we propose should provide needed information for this central area of macromolecular metabolism. Specific aims for this project are: 1) to purify and determine the physiological role and possible regulation of newly-discovered ribonucleases thought to be involved in tRNA processing and turnover; 2) to evaluate the diversity and role of phosphorolytic nucleases in RNA metabolism; 3) to identify and study the functional domains of TRNA inucleotidyltransferase; 4) to determine the importance of the -C-C-A terminus for TRNA function in vivo; 5) to study the role of lipids and membranes in aminoacyl-tRNA synthetase structure and function and to determine whether aminoacyl-tRNA is channeled in vivo; 6) to ascertain the functional. significance, if any, of the low molecular weight free form of arginyl-tRNA synthetase. In view of the central role of proteins in all cell functions, and the importance aminoacyl-tRNA formation for the specificity of this process, an understanding of the mechanism and regulation of this step is of great importance . Elucidation of these aspects of protein synthesis in normal cells is a prerequisite to any understanding of abnormal situations which may occur under pathological conditions.